Earlier this summer scientists from New England Biolabs in Beverly, Mass., announced they had discovered a new way to copy DNA. Their method, called helicase-dependent amplification (HDA), reportedly mimics in vivo DNA replication better than PCR does.1 Moreover, HDA can be conducted at a single temperature, thus obviating the need for thermocycling. In the future, perhaps the limitations of PCR – that it is reagent intensive, requires expensive equipment, is difficult to reproduce, and generally is not linear – will be surmounted by new technologies, HDA or otherwise.
TO TAQ OR NOT TO TAQ
In the beginning, there was
REAL-TIME RULES THE ROOST
Of the eight thermocyclers new to the market in 2004 and identified in this article, six are real-time instruments. Also known as quantitative PCR(QPCR) thermocyclers, these instruments monitor the accumulation of PCR product in real-time, automatically analyze data, and eliminate the need for many types of quantitative post-PCR processing.
According to product manager Andrew Felton of Applied Biosystems, Foster City, Calif., sample capacity is one of the most important features to consider when shopping for a QPCR cycler. Most QPCR thermocyclers on the market accommodate standard 96-well plates, but several companies are challenging that level of parallel processing. Applied Biosystems' high-throughput 7900 HT Sequence Detection System accommodates 384-well plates, for instance.
Woburn, Mass.-based Biotrove boasts that its OpenArray-based products will eventually be able to analyze 25,000 samples simultaneously, though it has yet to launch this technology. Scientists from 454 Life Sciences in Branford, Conn., announced in November 2003 the successful simultaneous amplification of approximately 300,000 discrete reactions using a novel platform called the PicoTiterPlate.2 Designed for whole-genome sequencing, the PicoTiterPlate is not yet commercially available.
On the other end of the spectrum, Salt Lake City-based Idaho Technology's new RAZOR instrument, launched in Spring 2004, is a battery-powered, 4-kg, stand-alone, real-time thermocycler with a 12-sample capacity. The company's HR-1 High-Resolution Melter, also launched this year, is a 2.65-kg, hand-held, single-sample device designed for gene scanning and mutation detection. Both devices are intended for defense personnel.
The next most important consideration after capacity, says Felton, is fluorescent dye flexibility. QPCR relies on a directly proportional relationship between the amount of accumulated double-stranded DNA (dsDNA) and the light signal emitted by dsDNA-bound fluorescent molecules or sequence-specific probes. The greater the number of dyes an instrument can accommodate, the more options an experimenter has. Multiplexing capacity, the instrument's ability to read multiple fluorescent signals in a single reaction tube, is another related consideration.
Other features to consider include: upgrade options (e.g., software), multiuser capabilities, robotic and automation capacity, PC-compatibility, run time, customer service, and price. La Jolla, Calif.-based Stratagene offers two QPCR systems, the primary differences being service and price. At $24,995, the more simply designed Mx3000 is considerably more affordable than the approximately $60,000 Mx4000, despite similar optical and amplification capacities. The Mx4000, however, comes with on-site installation, training, and repair service, whereas Mx3000 users must rely on the manual and return repair service. Stratagene offers Web-based seminars on the use of both instruments.
Comparable to Stratagene's Mx3000 is Applied Biosystems' newly launched entry-level 7300, which lists for $34,900 and comes with an optional future upgrade to high-speed cycling. If the 7300's fixed-ramp rates pose a limitation for users who have already optimized reaction conditions on older instruments, the 7500 model might be a better choice. At $42,500, the 7500 also includes upgraded software, five-color dye detection (as opposed to the 7300's four-color system), and variable-excitation wavelengths (the 7300 offers a single-excitation wavelength).
Other QPCR cyclers new to the market this year include the Quantica from Techne, Cambridge, UK; the InSyte from Biogene, near Cambridge, UK; and the LightCycler 2.0 from Roche, Mannheim, Germany. Also new this year, MJ Research of Waltham, Mass., offers the Chromo4 Four-Color Real-Time System, an optical block that converts any of that company's DNA Engine thermocyclers into a real-time system. (MJ Research's parent company, MJ GeneWorks, was acquired August 18 by Bio-Rad Laboratories of Hercules, Calif.)
TRADITION STANDS THE BETTER TEST OF TIME
Other new QPCR instruments may be on the market, but conventional thermocyclers still serve an invaluable basic research function. Traditional thermocyclers that entered the market in 2004 include the T3000 from Biometra in Göttingen, Germany, and the THEQ LifeCycler from MWG Biotech near Munich, Germany. The T3000 is a multiblock system (three independent blocks) featuring faster ramp times than its predecessor, the T3. (The maximum heating rate for the T3000 is 2.2°C/sec, compared to 1.5°C/sec for the T3. The maximum cooling rate for the T3000 is 2.0°C/sec, compared to 1.3°C/sec for the T3.) The T3000 also includes new, more user-friendly software.
The THEQ Life Cycler, which replaces MWG's old Primus line, is a server-satellite system that can expand up to as many as 48 networked units, all controlled by a single PC. According to company literature, THEQ instruments have the "largest color touch screen to date" and interface programming "so self-explanatory that the system can be operated virtually without ever having to consult the manual."
As with their real-time counterparts, sample capacity is an important consideration when choosing a conventional PCR instrument. Other considerations include block interchangeability (i.e., the ability to run 96 × 0.2 ml or 48 × 0.5 ml samples) and temperature gradient functionality. Temperature gradients allow the user to program multiple annealing temperatures in a single block, making for easier optimization of new primer sets.
Leslie A. Pray
Selected Suppliers of Thermostable DNA Polymerases
New England Biolabs
Roche Applied Science